Several temperate coliphages are known to express recombination functions. The int and xis genes of lambda, for example, specify site-specific recombination enzymes, while the reda and redb gene products, .lambda. exonuclease and beta protein, respectively, promote homologous recombination. The four genes are clustered in the lambda genome, and their roles in lambda physiology are known; the first two are required for prophage integration and excision, whereas the products of the red genes convert lambda monomers into packageable multimers.
Prophage P1 expresses a site-specific recombination function, Cre, which improves P1 plasmid maintenance by cutting and rejoining P1 chromosomes at the P1 lox sites (Hochman, L., et al. 1983. Virology 131:11-17; Sternberg, N., et al. 1980. Cold Spring Harbor Symp. Quant. Biol. 45:297-309; Sternberg, N., et al. 1986. J. Mol. Biol. 187:197-212). RecA is responsible for most P1 circularization, since Cre can circularize only those P1 molecules, about 20% of the total, which bear redundant lox sites. In recA mutants, however, Cre is essential for P1 lysogenization and lytic growth.
The lytic development of P1 prophage is blocked by the bacteriophage-encoded cI gene product. Lysogens carrying P1c1.100, a clts mutant, are stable at 32.degree. C. but are induced at temperatures above 39.degree. C. (Rosner, J. L. 1972. Virology 48:679-689). Several years ago, applicants observed that P1c1.100 lysogens displayed a phenotype related to recombination. Escherichia coli bearing the IS1 gal operon insertion, galTN102 (Jordan, E., et al. 1967. Mol. Gen. Genet. 100:296-306), revert to Gal.sup.+ at elevated frequencies when lysogenic for P1c1ts. This reversion probably results from recombination between the repeated 9-base-pair (bp) galT target sequences which flank the IS1 transposon, a process referred to as precise excision. Independently, Windle and Hays (Windle, B. E. and J. B. Hays. 1986. Proc. Natl. Acad. Sci. USA 83:3885-3889) noted that recombination between two close but nonoverlapping deletions in lacZ was likewise increased by P1c1.100 prophage. P1c1.100 did not appear to stimulate recombination within large regions of DNA homology (Windle, B. E. and J. B. Hays. 1986. Proc. Natl. Acad. Sci. USA 83:3885-3889). These observations suggested that P1 carries a recombination enhancement function (ref) which specifically stimulates recombination between small segments of homologous DNA flanked by nonhomologous sequences.
Little is known about microhomologous recombination. Excision of transposons does not depend upon transposase activity and is at least partially independent of the E. coli RecA pathway. Indeed, RecA does not efficiently promote pairing between homologous DNA segments 30 bp or less in extent (Argus, P., et al. 1986. EMBO J. 5:433-440). Several bacterial functions have been implicated in precise excision; mutations that stimulate the reaction have been mapped to recBC, uvrD, (mutU) mutH, mutL, mutS, mutD, ssb, and dam (Lundblad, V. and N. Kleckner. 1985. Genetics 109:3-19). Mutations in mutU and dam, as well as a variety of mutations which result in the accumulation of DNA replicative fragments, increase micro-homologous recombination in lacZ (Konrad, E. B. 1977. J. Bacteriol. 130:167-172). No demonstration of RecA-independent microhomologous recombination in vitro has been reported.
The subject invention describes the cloning and sequencing of the P1 ref gene and the overproduction of Ref in an expression vector system. Ref stimulation of precise excision is entirely dependent on the RecA pathway. In agreement with previously reported data (Windle, B. E. and J. B. Hays. 1986. Proc. Natl. Acad. Sci. USA 83:3885-3889), Ref does not play an indispensable role in the physiology of phage P1.
Applicants have found that Ref is useful for stimulating ligation of nucleic acid molecules.